Covalently closed circular or supercoiled DNA has become a topic of considerable interest in the field of molecular biology as a result both of its unique structural properties and its presence in a wide variety of biological locations. As a result of its appearance in the "life cycle" of various DNA and RNA tumor viruses, supercoiled DNA is especially relevant to current problems in viral oncogenicity. Using a wide variety of experimental approaches we and other investigators have concluded that superhelical DNA contains altered or interrupted secondary structure. In the case of SV40 and polyoma DNA the evidence strongly supports the appearance of unpaired bases at localized sites (1). In the last year we have explored a new chemical probe, a water soluble carbodiimide, for mapping the altered sites and developing this as a new experimental approach for structure-function relationships of superhelical DNA. In collaboration with investigatros at NIH listed in reference 1 we have mapped the binding of the above reagent in the restriction fragments where transcription is initiated in SV40 DNA E. coli RNA polymerase. Objectives: In light of the successful experiments with carbodiimide modification we set out to explore the following specific aims: 1) a more detailed mapping of the altered or interrupted secondary structure of SV40 by high specific activity carbodiimide binding coupled with enzymatic analysis; 2) an analysis of the role of the altered regions in transcription; 3) the continuation of the structure of circular DNA by full exploration of the reaction of carbodiimide and the development of other chemical probes; 4) the study of the functional capacity of chemically modified SV40 DNA. Bibliographic references: 1. Salzman, N.P., Lebowitz, J., Chen, M., Sebring, E. and Garon, C.F. Cold Spring Harbor Symposium on Quant. Biol. 39:209-218 (1974).